WNT Signaling, in Synergy with T/TBX6, Controls Notch Signaling by Regulating Dll1 Expression in the Presomitic Mesoderm of Mouse Embryos

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RESEARCH COMMUNICATION

1998; Jiang et al. 2000; Jouve et al. 2000; Aulehla et al. WNT signaling, in synergy 2003). The segmentation clock is closely linked to Notch with T/TBX6, controls Notch and WNT signaling activity. Most genes displaying cyclic activity encode components of the Notch pathway signaling by regulating Dll1 (Palmeirim et al. 1997; Forsberg et al. 1998; Jiang et al. 2000; Jouve et al. 2000); one other cyclic gene encodes expression in the presomitic the WNT pathway component Axin2 (Aulehla et al. mesoderm of mouse embryos 2003). Altered Notch signaling disrupts somite formation and patterning in Xenopus, zebrafish, and mouse Michael Hofmann,1 Karin Schuster-Gossler,2 embryos (Conlon et al. 1995; Hrabe de Angelis et al. Masami Watabe-Rudolph,2 Alexander Aulehla,1 1997; Jen et al. 1999; Holley et al. 2000; Jiang et al. 2000; Bernhard G. Herrmann,1,3,4 and Achim Gossler2,5 Sawada et al. 2000). Furthermore, mutations in some Notch pathway components, which lead to defects in 1Max-Planck-Institute of Immunobiology, Stübeweg 51, somitogenesis, also affect the expression of cyclic genes D-79108 Freiburg, Germany; 2Institute for Molecular Biology (del Barco Barrantes et al. 1999; Jiang et al. 2000; Jouve et OE5250, Medizinische Hochschule Hannover, al. 2000; Dunwoodie et al. 2002), indicating that Notch D-30625 Hannover, Germany signaling is essential for generating cyclic gene expression. Cyclic Lfng gene expression was shown in chick Notch signaling in the presomitic mesoderm (psm) is and mouse embryos to be essential for Lfng function critical for somite formation and patterning. Here, we (Dale et al. 2003; Serth et al. 2003). Disruption of WNT/ show that WNT signals regulate transcription of the ␤-catenin signaling also affects somitogenesis and cyclic Notch ligand Dll1 in the tailbud and psm. LEF/TCF fac- expression of Notch pathway components, whereas cy- tors cooperate with TBX6 to activate transcription from clic Axin2 expression is maintained when Notch signaling is impaired (Aulehla et al. 2003; Aulehla and Herr- the Dll1 promoter in vitro. Mutating either T or LEF/ mann 2004), suggesting that WNT acts upstream of Dll1 TCF sites in the promoter abolishes reporter gene Notch in the segmentation clock. However, the exact expression in vitro as well as in the tail bud and psm of molecular interplay between the various components of transgenic embryos. Our results indicate that WNT ac- these pathways is not fully understood. tivity, in synergy with TBX6, regulates Dll1 transcrip- T-box transcription factors as well as FGF and WNT tion and thereby controls Notch activity, somite forma- signaling are essential regulators of formation and differ- tion, and patterning. entiation or maintenance of paraxial mesoderm in mouse embryos. Mutations in T, Fgfr1, Wnt3a, and Tbx6 Received August 11, 2004; revised version accepted cause defects in formation and differentiation of paraxial September 13, 2004. mesoderm. Loss of T gene function leads to failure of axis development and arrested somite formation (Wilkinson The subdivision of the paraxial mesoderm into somites, et al. 1990; Herrmann 1995) most likely because of im- a metameric series of homologous subunits, is the most paired migration of mesodermal cells through the primi- obvious sign of segmentation in early vertebrate em- tive streak (Wilson et al. 1993). The loss of Wnt3a also bryos. During mouse embryogenesis, the first somites affects mesodermal cell migration (Yoshikawa et al. form in the posterior headfold region of the embryo 1997). In Wnt3a mutants presumptive paraxial meso- around embryonic day 7.75 (E7.75). Subsequently, new derm cells accumulate beneath the primitive streak and somites condense at regular intervals in a strict anterior- form neural tissues (Yoshikawa et al. 1997). The migra- to-posterior sequence from the unsegmented so-called tory defect in Wnt3a mutants might be due to reduced T presomitic mesoderm (psm) that lies caudally to the first function because T has been shown to be a direct target somites. Somite condensation progresses while con- of WNT3a signals in the paraxial mesoderm (Yamaguchi comitantly new paraxial mesoderm cells are being genet al. 1999). Embryos lacking Tbx6 produce presumptive erated caudally from the primitive streak and later from paraxial mesoderm, but the differentiation of these cells the tail bud elongating the embryo posteriorly. is impaired and supernumerary neural tubes form in- Somite formation is coupled to a molecular oscillator stead of somites (Chapman and Papaioannou 1998). In referred to as the segmentation clock, which has been embryos lacking Fgfr1 function, presomitic mesoderm revealed by the cyclic expression of genes in the psm. formation is impaired, leading to the complete absence Expression of cyclic genes is periodic such that one wave of somites and an excess of axial mesoderm (Yamaguchi of expression passes through the psm during the forma- et al. 1994). In chimeras, Fgfr1 mutant cells accumulate tion of one somite (Palmeirim et al. 1997; Forsberg et al. in the primitive streak, fail to colonize the paraxial mesoderm, and differentiate instead to neural tissue (Ciruna et al. 1997). Together these results suggest that WNT [Keywords: WNT signaling; Notch signaling; Dll1; somitogenesis; regulation] and FGF signaling in concert with T-box transcription 3Present address: Max-Planck-Institute for Molecular Genetics, Ihne- factors regulate the migration of mesodermal cells and straße 73, D-14195 Berlin, Germany. the choice between neural and paraxial mesodermal Corresponding authors. fates. 4E-MAIL [email protected]; FAX 49-30-8413-1229. 5E-MAIL [email protected]; FAX 49-511-532-4283. WNT signaling as a component of both the segmenta- Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/ tion clock and the regulatory network governing paraxial gad.1248604. mesoderm differentiation suggests that paraxial meso-

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Regulation of Dll1 by WNT and TBX6 derm formation and early patterning are tightly coupled. Further support for this idea comes from our analysis of the mouse rib-vertebrae (rv) mutation, which represents a hypomorphic Tbx6 allele causing reduced Tbx6 expression (Beckers et al. 2000b; Watabe-Rudolph et al. 2002). Reduction of Tbx6 mRNA leads to altered abundance and distribution of mRNAs encoding the Notch pathway components Dll1, Dll3, Lfng, and Notch1 in the presomitic mesoderm, and to somite defects similar to mutations in Notch pathway components (Beckers et al. 2000b). Notably, Dll1, which encodes a critical ligand for Notch in the psm, is severely down-regulated, and rv and a null allele of the Notch ligand Delta1 show nonallelic noncomplementation such that double heterozygotes have a mild “rv-phenotype” with complete penetrance (Beckers et al. 2000b). This raises the possibility that Tbx6 not only controls the differentiation of paraxial mesoderm, but might also directly activate expression of Dll1 (and other genes essential for somite patterning) and thereby regulate patterning in the psm. Here, we show that both T-box- and LEF/TCF-binding sites are essential for activity of the Dll1 promoter in the tailbud and presomitic mesoderm, suggesting that T/TBX6 and WNT signaling directly and synergistically Figure 1. Expression of Dll1 or Tbx6 in T and Tbx6 mutant em- regulate Dll1 transcription in the tailbud and presomitic bryos. In situ hybridization of E8 (a,b,e,f) and E8.5 (c,d,g–j) wild-type (a,c,e,g,i) and homozygous T (f,h,j) and Tbx6 (b,d) mutant embryos mesoderm. Thus, WNT signals not only regulate meso- with Dll1 (a–h) and Tbx6 (i,j) probes. Dll1 expression was signifi- derm formation upstream of T and Tbx6, but also regu- cantly down-regulated in E8 and not detected in E8.5 T and Tbx6 late patterning in the psm cooperatively with transcrip- mutant embryos. In E8.5 T mutant embryos Tbx6 expression was tion factors that are themselves targets of WNT activity. significantly reduced.

data suggest a cascade of factors involved in the control Results and Discussion of Dll1 expression. The reduction of Dll1 transcripts in T mutant em- During our analysis of the mouse rv mutation we no- bryos, and their reduction and subsequent loss in Tbx6 ticed that Dll1 expression is significantly reduced in the mutant embryos could indicate that Dll1 is a direct tar- presomitic mesoderm of rv mutant embryos (Beckers et get of these transcriptional regulators. To explore this al. 2000b). rv is a hypomorphic allele of the Tbx6 gene possibility further, we analyzed the sequence of the pro- (Watabe-Rudolph et al. 2002), which encodes a T-box moter region of Dll1 that was previously shown to con- transcription factor specifically expressed in the primi- tain sequences sufficient to drive heterologous gene ex- tive streak/tailbud and presomitic mesoderm (Chapman pression in the psm for potential T-box-binding sites. In et al. 1996). To address how the complete loss of Tbx6 this genomic region, which comprises 4.3 kb upstream of function affects Dll1, we analyzed Dll1 expression in the translational start site, nine putative T-binding sites embryos carrying a targeted null allele of Tbx6. Embryos were identified (Fig. 2A,B). Six of these sites (T2–T7) are lacking Tbx6 function have five to seven irregular located within a 1.4-kb “msd” fragment, which drives somites in the prospective hindbrain region, but more heterologous gene expression robustly throughout the posteriorly they form ectopic neural tubes instead of par- psm and in newly formed somites (Beckers et al. 2000a). axial mesoderm (Chapman and Papaioannou 1998). Dll1 One T site (T1) is present at the distal end of the 4.3-kb expression in Tbx6-null mutant embryos was severely fragment; two other sites, T8 and T9, are in the proximal down-regulated on E8 (Fig. 1b), and Dll1 transcripts were region in exon 1 close to the ATG. In addition to the not detected on E8.5 (Fig. 1d) prior to the overt loss of potential T-binding sites, we found eight LEF/TCF-bind- paraxial mesoderm. This suggested that Tbx6 is required ing sites in the 4.3-kb Dll1 promoter region, four of to maintain Dll1 expression in the presomitic meso- which are located in the msd fragment (Fig. 2A,B), raising derm. Brachyury (T) is expressed in the primitive streak the possibility that WNT activity in addition to T/TBX6 and nascent mesoderm, overlapping with Tbx6. T is controls expression of Dll1 in the tailbud and psm. functionally upstream of Tbx6, since it is required for To address whether T, TBX6, and LEF/TCF proteins mesoderm formation (Herrmann et al. 1990), while Tbx6 can activate transcription from the msd promoter frag- acts in the maintenance and/or differentiation of par- ment, we generated a reporter construct, which ex- axial mesoderm (Chapman and Papaioannou 1998). presses luciferase fused to the Dll1 minimal promoter Similar to Tbx6 mutants, E8–E8.5 embryos lacking T (Beckers et al. 2000a) under the control of msd (msd- showed severely reduced or no Dll1 expression in the wtluc), cotransfected this construct together with ex- psm (Fig. 1f,h). These data raised the question whether pression vectors containing cDNAs encoding T, TBX6, Tbx6 expression depends on T. Indeed, we found that or TCF1E into COS7 cells, and determined luciferase Tbx6 is severely down-regulated in embryos lacking T activity. Full-length T and TBX6 resulted only in very (Fig. 1j). The presence of T sites in introns 1 and 5 of weak transactivation (data not shown), most likely be- Tbx6 (data not shown) supports the supposition that cause of a requirement for unknown cofactors. There- Tbx6 is a direct target of T. The combined expression fore, we generated fusions between the DNA-binding do-

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no synergistic activation of luciferase activity was ob- served in contrast to cotransfections of TBX6VP16 and LEF1 either with or without ␤-catenin (Fig. 3B). Thus, synergism between TBX6VP16 and LEF/TCF appears to depend on ␤-catenin, suggesting that in the presomitic mesoderm Dll1 is directly controlled byTBX6 and WNT signaling. To demonstrate that the T and LEF/TCF sites mediate transactivation from msd, T sites or LEF/TCF sites or a combination of these sites in msdwtluc were mutated, and mutated constructs were analyzed for luciferase expression. Mutation of all T sites except for the weakly conserved site T2 (msd5xTmut-luc, Fig. 2A,B) abolished the synergistic activation of luciferase by TBX6VP16 and TCF1E (Fig. 3C). In contrast, mutation of all four LEF/ TCF sites in msd (msd4xLmut-luc) did not affect the cooperative stimulation of transcription by TBX6VP16 and TCF1E (Fig. 3C), suggesting that the LEF/TCF site in the Dll1 minimal promoter fragment close to the ATG (L8, Fig. 2A) might be sufficient to compensate in this assay for the loss of LEF/TCF sites in msd. Indeed, no synergistic activation of luciferase expression was obtained from a construct (msd5xLmut-luc) in which this site was mutated in addition (Fig. 3C). Mutation of five T sites and the four LEF/TCF sites in msd (msd5xT4xLmut-luc) also abolished the synergistic activation. The loss of cooperative transactivation from msd5xTmut-luc, which still contains two potential T sites, T8 and T9, close to the ATG (Fig. 2A) suggests that these sites do not contribute to activation by T-box transcription factors under these conditions. Figure 2. T-box and LEF/TCF sites in the Dll1 promoter and re- To address the significance of the potential T and LEF/ porter constructs. (A) Schematic representation of the Dll1 promoter region extending 4.3 kb upstream of the ATG. The position of potential LEF/TCF sites (triangles) and T sites (circles) is indicated. H1 and H2 refer to the regions of homology detected between the zebrafish deltaD and mouse Dll1 promoters, which direct gene expression into the central nervous system (Beckers et al. 2000a). msd indicates the region directing gene expression in the paraxial mesoderm (Beckers et al. 2000a). (B) Sequences of the potential T and LEF/TCF sites in the Dll1 4.3-kb promoter region. Arrows indicate the orientation of the sites, and the consensus binding sites (cons) for LEF/TCF (Giese et al. 1992; van de Wetering and Clevers 1992) and T-box (Kispert et al. 1995; Kusch et al. 2002) factors are shown below.(C) Schematic representation of mutant reporter gene constructs used in this study. The mutated sites are marked and indicated above the constructs. mains of these proteins and the VP16 transactivator domain (TVP16 and TBX6VP16, respectively) for further analyses. Either transcription factor alone or in combination with ␤-catenin stimulated luciferase activity two- to threefold (Fig. 3A). Coexpression of TVP16 and TCF1E did not enhance luciferase activity above these levels. However, coexpression of TBX6VP16 and TCF1E stimulated a ninefold increase of luciferase activity (Fig. 3A), suggesting that TBX6 and TCF1E cooperate to activate transcription from msd. Coexpression of exogenous Figure 3. Synergistic activation of transcription from the msd pro- ␤-catenin with TBX6 and TCF1E did not further stimu- moter fragment by TBX6 and LEF/TCF. Relative luciferase activity in COS7 cells transfected with reporter gene plasmids and combi- late luciferase activity, which is most likely because of ␤ nations of expression vectors as indicated. Luciferase activity was endogenous -catenin present in COS7 cells (data not normalized to activity obtained with the wild-type msd reporter shown). To demonstrate that the synergistic effect of plasmid alone. (A) TBX6VP16 and TCF1E activate the msd promoter TBX6VP16 and LEF/TCF factors is ␤-catenin dependent, synergistically. (B) Synergism between TBX6VP16 and LEF1 de- we cotransfected COS7 cells with TBX6VP16, ␤-catenin, pends on the ␤-catenin-binding domain of LEF1. (C) Both T and and LEF1 or a truncated version of LEF1 (LEF1⌬7–89) LEF/TCF sites are critical for synergistic activation. For comparison ␤ of luciferase activity between different reporter constructs, activity that is defective for binding of -catenin and acts as a of the mutant promoter constructs was related to the activity ob- dominant-negative protein (Korinek et al. 1997; Hovanes tained with the msd wild-type construct, whose activity was set to et al. 2001), respectively. In the presence of LEF1⌬7–89 one.

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Regulation of Dll1 by WNT and TBX6

ers et al. 2000a). When five of the six T sites in msd were mutated (T3–T7; msd5xTmut-lacZ, Fig. 2C), highly variable lacZ expression in the somites but no expression in the psm was detected (n = 7) (Fig. 4B, panels b,e). Collec- tively, these results demonstrate that T sites in the Dll1 promoter are critical for Dll1 expression in the tailbud and presomitic mesoderm, strongly supporting the idea that T and/or TBX6 are direct regulators of Dll1 in vivo. Similar to msd with mutated T sites, mutations in all four LEF/TCF sites in the msd promoter (msd4xLmut- lacZ, Fig. 2C) abolished lacZ expression in the psm of transgenic embryos but retained variable somitic expression (n = 11) (Fig. 4B, panels c,f). The combination of mutated T and LEF/TCF sites (msd 5xT4xLmut-lacZ, Fig. 2C) gave similar results with additional variable ectopic lacZ expression in parts of the central nervous system (n = 10) (Fig. 4B, panels d,g). Variable ectopic expression and apparent augmented somitic expression most likely reflect position effects. In addition, loss of LEF/TCF mediated repression in the absence of WNT activity (Roose et al. 1998) might contribute to ectopic or augmented expression from transgene constructs with Figure 4. Reporter gene expression in transgenic embryos. Repre- mutated LEF sites. Consistent with the results of our in sentative examples of ␤-galactosidase-stained transgenic embryos vitro studies, these analyses indicate that binding sites obtained after DNA microinjection. (A) Staining patterns in em- both for T-box and LEF/TCF transcription factors and bryos carrying the wild-type (panel a) and mutant (panels b–d) 4.3-kb promoter constructs. Note that mutations in either T1 (panel b)or thus binding of both factors are simultaneously required T5 (panel c) abolished ␤-galactosidase expression specifically in the in the msd enhancer to direct transcription in the psm. tailbud (arrowheads). Arrows point to the most recently formed This strongly suggests that T-box transcription factors somites. Insets show higher-magnification pictures of the tails. (B) and WNT activity cooperatively regulate Dll1 expres- Staining patterns in embryos carrying the wild-type (panel a) and sion in vivo in the tailbud and psm. Consistent with this mutant (panels b–g) msd promoter constructs. Mutation of either T interpretation, a targeted mutation of Lef1, which inter- sites (panels b,e), or LEF/TCF sites (panels c,f), or both (panels d,g) ␤ completely abolished ␤-galactosidase in the presomitic mesoderm, feres with -catenin-dependent activation by LEF/TCF which expresses high levels of ␤-galactosidase in wild-type msd– proteins (Galceran et al. 2000), causes down-regulation lacZ transgenic embryos. of Dll1 in the psm, and somite patterning defects (Gal- ceran et al. 2004). Synergistic activation of the msd promoter in COS TCF sites for Dll1 expression in the presomitic meso- cells by TBX6VP16 and LEF/TCF but not by TVP16 and derm of mouse embryos, we generated various lacZ re- LEF/TCF (Fig. 3A) could indicate that also in vivo TBX6 porter constructs with the 4.3-kb promoter fragment rather than T directs Dll1 expression. Thus, the reduc- that carried point mutations in individual or multiple T tion or loss of Dll1 expression in T−/− embryos (Fig. 1f,h) sites, as well as with the msd promoter carrying muta- could be an indirect effect of the severe down-regulation tions in the T and LEF/TCF sites as before (Fig. 2C), and of Tbx6 in these mutants (Fig. 1j). Alternatively, T might analyzed lacZ expression in embryos after DNA micro- specifically interact with site T1, either alone or in com- injection into zygotes (Fig. 4). The 4.3-kb promoter frag- bination with (an)other more proximal T site(s), and thus ment recapitulates many aspects of early Dll1 expression might contribute to expression of Dll1 in the tailbud. and gives rise to lacZ expression in the tailbud, presomitic mesoderm, and somites as well as in the central nervous system (Beckers et al. 2000a). We have previously shown that deletion of the most distal region of the 4.3-kb promoter resulted in loss of transgene expression in the tailbud (Fig. 7 in Beckers et al. 2000a). This region contains the T site T1. When T1 was mutated (4.3T1mut-lacZ, Fig. 2C), lacZ expression in the tailbud was abolished (n = 8) (Fig. 4A). Similarly, when T5, which resides in msd and is very similar to the consensus T-binding site (4.3T5mut-lacZ, Fig. 2A,B), was mutated, no lacZ expression in the tailbud was detected Figure 5. Proposed regulatory network directing Dll1 expression in (n = 21) but psm expression was maintained, suggesting the tailbud and presomitic mesoderm. WNT signals induce meso- that each of these T sites is essential for Dll1 expression derm formation and Brachyury expression, whose function is essen- specifically in the tailbud. Mutation of eight of the nine tial for migration of mesodermal cells through the primitive streak. T sites in the promoter, excluding the weakly conserved T acts upstream of Tbx6, potentially as direct activator, and Tbx6 is site T2 (4.3 8xTmutlacZ, Fig. 2C), resulted in the com- required to maintain paraxial mesoderm. Both T and Tbx6 regulate plete loss of expression in the tailbud, psm, and newly Dll1 in the tailbud and in the presomitic mesoderm, respectively. WNT signals acting through the canonical Wnt pathway control formed somites (n = 5) (Fig. 4A, panel d). The msd frag- Dll1 expression in the paraxial mesoderm synergistically with ment drives heterologous gene expression robustly TBX6. (Bold arrows) Proven direct regulation; (dashed arrows) ge- throughout the psm and in newly formed somites (Beck- netically upstream.

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Indeed, recombinant T protein can bind weakly to T1 P-40, and 10% glycerol on ice for 20 min. Firefly luciferase and ␤-galac- but effectively to a synthetic DNA fragment containing tosidase activities were measured in a Luminoskan Ascent luminometer T1 and the highly conserved site T5 (data not shown), (Thermo Labsystems). The reporter gene activities shown are average supporting this possibility. values, obtained from at least four independent experiments. Canonical WNT signals control mesoderm formation ␤ and T expression (Takada et al. 1994; Liu et al. 1999; DNA microinjection and -galactosidase staining Transgenic embryos were produced by pronuclear injections of linearized Yamaguchi et al. 1999; Arnold et al. 2000). Our data sug- DNA constructs into fertilized eggs derived from strain FVB/N according gest that WNT activity, in addition to regulating genes to standard techniques, dissected at the indicated stages, and processed required for mesoderm formation, directly controls the for ␤-galactosidase staining as described (Beckers et al. 2000a). expression of genes that are critical for patterning of the paraxial mesoderm (Fig. 5), and further support our pre- vious suggestion that WNT signaling controls the seg- Acknowledgments mentation process (Aulehla et al. 2003). By regulating We thank Manuela Scholze for excellent technical assistance, A. Hecht Dll1 expression, WNT indirectly controls the signaling and R. Kemler for reagents, V. Papaioannou for Tbx6 mutant mice, and J. activity of Notch. Thus, WNT activity regulates mul- Galceran and R. Grosschedl for communicating results prior to publica- tiple aspects of mesoderm development not only by act- tion. This work was supported by grants of the Deutsche Forschungsge- ing high up in the genetic hierarchy that governs meso- meinschaft to B.G.H. (SFB592) and A.G. (Go449/5-2). derm formation but also by directly cooperating with direct or indirect targets of its own activity. References

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Regulation of Dll1 by WNT and TBX6

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GENES & DEVELOPMENT 2717 Downloaded from genesdev.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press

WNT signaling, in synergy with T/TBX6, controls Notch signaling by regulating Dll1 expression in the presomitic mesoderm of mouse embryos

Michael Hofmann, Karin Schuster-Gossler, Masami Watabe-Rudolph, et al.

Genes Dev. 2004, 18: Access the most recent version at doi:10.1101/gad.1248604

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